The overall goal is to understand the molecular mechanisms that mediate the reactions of DNA replication. Structure studies of the individual proteins, their functional sub-assemblies, and the replisome itself combined with genetic and biochemical analyses will define the protein-protein interactions that coordinate the steps. Studies focus on the replication system derived from phage T7 since its chromosome is replicated in a manner characteristic of those of more complex systems, but by a small number of proteins, most encoded by the phage. The crystal structure of T7 DNA polymerase complexed to a primer-template, a deoxynucleoside triphosphate, and its processivity factor, E. coli thioredoxin, provides insight into polymerization and suggests future studies. The mechanism of processivity conferred by the thioredoxin-thumb subdomain will be pursued by identifying contacts of this subdomain with the DNA and other residues on the polymerise and by examining the movement of the complex on DNA. The gene 4 protein provides helicase and primase activities at the replication fork. The two activities will be studied independently using cloned domains that encode each. The domain responsible for the formation of the functional helicase hexamer will be identified and its interaction with both strands of a duplex DNA substrate will be characterized. Sequence-specific recognition of single-stranded DNA by the primase domain will be studied by determining the role of the CyS4 zinc motif and by identifying the nucleotide binding sites for primer synthesis. The interaction of the helicase and primase with the polymerase will be dissected by use of altered proteins and by suppressor mutation analysis. The T7 gene 2.5 single-strand binding protein interacts with all the T7 replication proteins and is essential for coordination of reactions at a replication fork. The role of the gene 2.5 protein dimer in homologous base-paining and in protein-protein interactions will be examined and its binding to single-stranded DNA will be characterized. The interaction of gene 2.5 protein with DNA polymerase and gene 4 protein will be pursued through the use of in vitro mutagenesis and cross-linking studies. A mini-circle replication system in which leading and lagging strand synthesis are coupled and the lagging strand polymerase operates processively will be used to dissect protein interactions at the fork, particularly those involving gene 2.5 protein. The mechanism by which a replication loop is formed and its relationship to Okazaki fragment size will be pursued. The mini-circle replication system will be used to examine replication phenomena such as the formation of expansion of trinucleotide repeats.